PCR Primer Design

Description

This third edition provides new and updated chapters on design PCR primers for successful DNA amplification. Chapters are divided into seven parts, including primer design strategies for quantitative PCR, genotyping, multiplex PCR, in silico PCR primer design, and primer design to identify plant and animal viruses. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, PCR Primer Design, Third Edition aims to be useful for various fields of molecular biology, including biotechnology, molecular genetics, and recombinant DNA technology. Dedication Preface Contents Contributors Part I: Primer Design for Genotyping Chapter 1: The Significance of PCR Primer Design in Genetic Diversity Studies: Exemplified by Recent Research into the Genetic... 1 Introduction 2 Research Tasks 3 Sample Collection and DNA Extraction 4 Microsatellite Markers 4.1 Isolation, Primer Design, and Amplification 4.2 Paternity Testing 4.3 Genetic Diversity Studies 5 SNP Markers 5.1 SNP Discovery from EST Sequences 5.2 SNP Discovery and Genotyping by Diversity Array Technology 5.3 Genetic Diversity Studies 6 Notes References Chapter 2: Enhancing Cohort PASA Efficiency from Lessons Assimilated by Mutant Genotyping in C. elegans 1 Introduction 2 Advances in Primer design Strategies 2.1 Enhancing Specificity and Discriminatory Potential of Allele-Specific Primers 2.2 Enhancing Sensitivity of PASA-Based Approach 2.3 Web-Based Applications for Primer Design 3 Advances in Detection Techniques of PASA 3.1 Increased Sensitivity of Detection 3.2 Reduced Cost and Time 4 Cohort PASA, a Case Study in C. elegans 4.1 Mechanism Involved in Cohort PASA 4.2 Primer Design Strategy 4.3 Genomic DNA Extraction 4.4 Determining Experimental Annealing Temperature for Cohort PASA 4.5 Differentiating Homozygotes from Heterozygotes 5 Conclusions References Chapter 3: Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques 1 Introduction 2 General Requirements 2.1 In Silico Design 2.2 Primer Validation 3 Primer Design for Allele Enrichment in PCR-Based Methods 3.1 Method Principle 3.2 Design Considerations 3.3 Enhanced Approaches 4 Primer Design for Allele Suppression in PCR-Based Methods 4.1 Method Principle 4.2 Design Considerations 4.3 Enhanced Approaches 5 Primer Design in Isothermal Methods 5.1 General Considerations 5.2 Allele-Specific RPA 5.3 Blocked RPA 5.4 Allele-Specific LAMP 6 Examples of Applications 6.1 Example 1 6.2 Example 2 6.3 Example 3 6.4 Example 4 6.5 Example 5 7 Conclusions References Chapter 4: Detection of Rubella Virus by Tri-Primer RT-PCR Assay and Genotyping by Fragment RT-PCR 1 Introduction 2 Materials 2.1 RNA Preparations 2.2 RT-PCR 2.3 Primers for Diagnostic PCR 2.4 Primers for Genotyping 2.4.1 Primer Pair-1 2.4.2 Primer Pair-2 2.5 Gel Electrophoresis 2.6 Sanger Sequencing 2.7 Sequence Analysis 3 Methods 3.1 Clinical Specimen 3.2 RNA Preparations 3.3 Assay Controls 3.3.1 Positive Controls 3.3.2 Negative Controls Water Controls Extraction Controls 3.4 For Diagnostic PCR 3.4.1 Rubella Genotyping 3.4.2 Rubella Genotyping (Using the Superscript III Kit) 3.4.3 Rubella Genotyping (Using the Qiagen One Step RT-PCR Kit) 4 Conclusion 5 Notes References Chapter 5: Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentic... 1 Introduction 2 Materials 2.1 Sequence Retrieval and Design of Primer/Probe Systems 2.2 In Silico Evaluation 2.3 Evaluation by Real-Time PCR 3 Methods 3.1 SNP Search and Sequence Retrieval for Primer Design 3.2 Sequence Retrieval in Case No SNP Has Been Found in Literature 3.3 Design of a Primer/Probe System 3.4 Introduction of One Mismatch Base 3.5 In Silico Evaluation 3.6 Evaluation by Real-Time PCR 3.7 Introduction of Two Mismatch Bases 3.8 Introduction of Three Mismatch Bases 3.9 Optimization of Real-Time PCR Conditions 4 Notes References Chapter 6: Primer Design for the Analysis of Closely Related Species: Application of Noncoding mtDNA and cpDNA Sequences 1 Introduction 2 Materials 2.1 PCR Evaluation 2.2 Sequence Comparison and Primer Design 3 Methods 3.1 DNA Isolation and PCR Evaluation 3.2 In Silico Evaluation 3.3 Optimization of PCR Conditions 4 Notes References Chapter 7: Designing PCR Primers for the Amplification-Refractory Mutation System 1 Introduction 2 Methods 2.1 Examination of Primers Using Integrated DNA Technology (IDT) Website 2.2 Examination of Specificity of the Designed Primers References Part II: Primer Design for Genome-Wide Identification of Specific Regions Chapter 8: Validation of Circular RNAs by PCR 1 Introduction 2 Materials 2.1 Divergent and Full-Length Primer Design 2.2 RNase R Treatment and RT-qPCR 2.3 Purification and Sanger Sequencing of PCR Products Amplified with Divergent and Full-Length Primers 3 Method 3.1 CircRNA PCR with Divergent Primers 3.1.1 Designing Divergent Primers 3.1.2 RNase R Treatment and cDNA Synthesis 3.1.3 Identification of circRNA by PCR with Divergent Primer 3.1.4 Validation of Circularity by Quantitative (Q)PCR 3.2 CircRNA PCR with Full-Length Primers 3.2.1 Full-Length circRNA Primer Design 3.2.2 Rolling Circle Reverse Transcription to Generate Full-Length cDNA 3.2.3 CircRNA PCR Using Full-Length Primers 3.2.4 Identification of Full-Length Spliced Sequence of circRNA 4 Notes References Chapter 9: Primer Designing for Amplifying an AT-Rich Promoter from Arabidopsis thaliana 1 Introduction 2 Materials 2.1 Plant Material and Growth Conditions 3 Methods 3.1 Guidelines for Primer Designing 3.1.1 Primer Length 3.1.2 GC Content and GC Clamp 3.1.3 Melting Temperature (Tm) 3.1.4 Annealing Temperature 3.1.5 Secondary Structures 3.1.6 Specificity Check 3.2 Primer Designing for Promoter Region 3.3 Genomic DNA Isolation 3.4 PCR Conditions 3.5 Amplicon Sequence Analyses 4 Notes References Part III: Primer Design for Multiplex PCR. Multiplex Chapter 10: PLASmid TAXonomic PCR (PlasTax-PCR), a Multiplex Relaxase MOB Typing to Assort Plasmids into Taxonomic Units 1 Introduction 2 Materials 2.1 Solutions (See Notes 1 and 2) 2.2 Equipment 3 Methods 3.1 Preparation of the PlasTax-PCR DNA Template 3.2 PlasTax-PCR for Enterobacterial Plasmids 4 Notes References Chapter 11: Multiplex PCR Design for Scalable Resequencing 1 Introduction 2 Materials 2.1 PCR Primer Ordering 2.2 PCR Reagents 3 Methods 3.1 Primer Design and Resuspension 3.2 Singleplex Screening and Quality Control 3.3 Multiplex Screening and QC 3.4 Multiplex Amplification of Target Samples and Library Preparation 4 Notes References Part IV: Primer Design for qPCR Chapter 12: Identification of Gene Copy Number in the Transgenic Plants by Quantitative Polymerase Chain Reaction (qPCR) 1 Introduction 2 Materials 2.1 Plant Material 2.2 Material for Genomic DNA Isolations 2.3 Genomic DNA Isolation 2.4 Genomic DNA PCR 2.5 Primer Design 2.6 Genomic DNA qPCR 3 Methods 3.1 Plant Material 3.2 Isolation of Genomic DNA of Arabidopsis Seedlings 3.3 Genomic DNA PCR 3.4 Primer Design for TCN Analysis 3.5 qPCR for TCN Analysis 3.6 Quantification of Copy Number 4 Notes 5 Conclusion References Chapter 13: qPrimerDB: A Powerful and User-Friendly Database for qPCR Primer Design 1 Introduction 2 Materials 2.1 Computer and Websites 2.2 DNA Sequences and Gene ID 3 Methods 3.1 Accessing Website 3.2 Primer Search 3.2.1 Search by Gene ID 3.2.2 Search by Sequence BLAST 3.3 Checking Primers 4 Notes References Part V: Primer Design for Identification of Plant and Animal Viruses Chapter 14: PCR Primer Design for the Rapidly Evolving SARS-CoV-2 Genome 1 Introduction 2 Materials 2.1 Prepare SARS-CoV-2, Human and Influenza Virus Genome Database 2.2 Install MicroGMT, Primer3, MFEprimer-3.0, and Other Software 3 Methods 3.1 Identify the Mutations Compared to the Reference Genome 3.2 Design Primers 3.3 Evaluate Primers 3.4 Re-evaluate Primers when Database Growing 4 Notes References Chapter 15: Universal Primers for Detection of Novel Plant Capsid-Less Viruses: Papaya Umbra-like Viruses as Example Abbreviations 1 Introduction 2 Materials 2.1 Software Tools and Web-Based Applications 3 Methods 3.1 Sequence Download 3.1.1 Searching and Downloading by Accession Numbers 3.1.2 Searching and Downloading by Keywords 3.1.3 Searching and Downloading by Homology 3.2 FASTA File Editing 3.3 Multiple Sequence Alignment 3.4 Identification of Conserved/Variable Regions 3.5 Phylogenetic Inference for Grouping 3.6 Primer Design and Analysis 3.7 Checking for Specificity and Cross-Reactivity of Primers: In Silico PCR 4 Notes References Part VI: Use of Software for Primer Design Chapter 16: A Guide to Using FASTPCR Software for PCR, In Silico PCR, and Oligonucleotide Analysis 1 Introduction 2 Software, General Information 3 The Interface 3.1 Inputs to FastPCR 3.2 Program Output 3.3 Sequence Entry 4 The PCR Primers or Probe Design Analyze Options 4.1 PCR Primer Design Generalities 4.2 Melting Temperature Calculation 4.3 Primer Quality (Virtual PCR Efficiency) Determination 4.4 Hairpin (Loop) and Dimer Formation 4.5 Secondary Nonspecific Binding Test; Alternative Amplification 5 Methods 5.1 Execution Selected Task 5.2 PCR Primer Design Options 5.3 Examples for Primer Selection Region 5.4 PCR Primer Design 5.5 Multiplex PCR Primer Design 5.6 In Silico PCR 5.7 Primer(s) Analyses 6 Availability References Part VII: Primer Design for Newer PCR Approaches Chapter 17: Pyrosequencing Primer Design for Forensic Biology Applications 1 Introduction 2 Materials 2.1 Websites and Software 2.2 Obtaining a DNA Sequence for the Region to be Sequenced 3 Methods 3.1 Design of Pyrosequencing Primers 3.2 Evaluating Nonspecific Priming by NCBI BLAST Nucleotide 3.3 Performing In Silico PCR 3.4 Obtaining Pyrosequencing Primer Reagents 4 Notes References Chapter 18: Phosphate-Methylated Oligonucleotides as a Novel Primer for PCR and RT-PCR References Index